To link to the entire object, paste this link in email, IM or documentTo embed the entire object, paste this HTML in websiteTo link to this page, paste this link in email, IM or documentTo embed this page, paste this HTML in website

Dynamics of amelogenin self-assembly during in vitro proteolysis by Mmp-20

DYNAMICS OF AMELOGENIN SELF-ASSEMBLY DURING IN VITRO PROTEOLYSIS BY MMP-20
by
Ruiwen Ma
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CRANIOFACIAL BIOLOGY)
May 2011
Copyright 2011 Ruiwen Ma

Amelogenin self-assembly and its stepwise processing are crucial for the enamel biomineralization. The removal of highly conserved (A- and B-) domains affects amelogenin self-assembly, resulting in enamel defect in transgenic and knock-in mice. We hypothesized that the amelogenin self-assembly can be altered by its stepwise proteolysis by Mmp-20, and the changes in self-assembly will eventually affect the structural organization of the forming enamel matrix, apatite nucleation, and mineralization. To examine the hypothesis, the full-length recombinant pig amelogenin (rP172), and a recombinant analogue (rP148) representing the most stable proteolytic product (the “20k”) were used. Recombinant rpMmp-20 was used to digest rP172 at pH 8.0 and pH 7.4, at temperature 37 °C, with substrate: enzyme ratios of 100:1 and 200:1. The proteolysis progress and particle size distribution during proteolysis were monitored by SDS-PAGE and dynamic light scattering.; At pH 8.0, 37ºC, SDS-PAGE revealed that, a major product (2-148 segment) was detected after 1 hour of proteolysis, and was accumulated until up to 20 hours; when the majority of the substrate was cleaved. This major product was confirmed to be the analogue of native “20k” amelogenin. Mass spectrometric analysis confirmed the presence of the Tyrosine Rich Amelogenin Polypeptide (TRAP) and other small proteolytic fragments. Analysis by dynamic light scattering revealed that rP172 possessed a distribution of particles with RH of 13.49 nm. During the first ten minutes of rP172 proteolysis small particles of 3.8 nm appeared in the solution. After the first hour of rpMmp-20 action, in addition to the 3.8 nm and 19.7 nm particles, large mono-dispersed assemblies with RH of 103.8 nm were also detected. Following 2 hours of proteolysis, the sizes of the large assemblies increased to about 125.7 nm. DLS analysis of a 1:1 mixture of recombinant rP172 and rP148 demonstrated a significant shift in particle size distribution when compared to the individual components: rP172 and rP148 alone. Using TEM, we detected monodispered spherical particles (~16nm diameter) formed on the TEM grid of rP172 at pH 8.0. After rpMmp-20 digestion, aggregation of the spherical particles was initiated around 1 hour, and chain formation was evident during 2nd to 4th hours, chain-like structures formed around 4 hours and curled after 20 hours. A general decrease in diameters was also observed after proteolysis. While the pure amelogenin samples (rP172, rP148) did not show any signs of chain formation after 24 hours incubation.; At pH 7.4, SDS-PAGE revealed that the proteolysis proceeded during the first hour and slowed down after 2 hours comparing to that at pH 8.0. In DLS analysis, rP172 possessed a major particle size distribution of RH at 28.3 nm. During the first ten minutes of proteolysis, small particles of 5.7 nm appeared in the solution. After the first hour of rpMmp-20 action, in addition to the 2.6 nm and 11.5 nm particles, large assemblies with RH of 45.3 nm were also detected. Following 10 hours of proteolysis, the sizes of the large assemblies increased to about 151.1 nm. Large aggregates (≥7000 nm) presented throughout the proteolysis, with a gradual decrease in their percentage mass. Under TEM observation, trends in nano-chain assembly was demonstrated similar to that mentioned at pH 8.0 proteolysis, but the appearance of chain-assembly occurred earlier than in the case of pH 8.0 (i.e. after 2 hours). No sign of chain formation was observed after incubating rP172 for 4 days.; These results support the hypothesis that the stepwise cleavage of amelogenin by Mmp-20 alters the dynamic of amelogenin self-assembly promoting the formation of chain-like structures. And there is a co-assembly between the full-length amelogenin and its proteolysis products, mainly the 2-148. It is proposed that such proteolysis activities will control the structural organization of forming enamel matrix and eventually the process of elongated apatite growth.

DYNAMICS OF AMELOGENIN SELF-ASSEMBLY DURING IN VITRO PROTEOLYSIS BY MMP-20
by
Ruiwen Ma
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CRANIOFACIAL BIOLOGY)
May 2011
Copyright 2011 Ruiwen Ma